The long-term objective of this project is to continue development of probes and methodologies for analysis of individual cells by flow or image cytometry applicable to tumor diagnosis and prognosis and for studies of apoptosis, cell proliferation and antitumor drug screening. The specific aims represent the continuation of four recently initiated projects: (1) DNA damage: Histone H2AX phosphorylation reveals the site of DNA double strand breaks (DSBs). We will: (a) design methods to distinguish antitumor drug induced (DI), primary DSBs from apoptosis-associated, secondary DSBs; (b) assess the kinetics of H2AX phosphorylation and dephosphorylation as a function of drug dose and treatment duration; (c) correlate the extent of H2AX phosphorylation with early events of apoptosis; (d) study how the rate of DNA replication and/or transcription affects the incidence of DI DSB; (e) explore whether phosphorylation of H2AX can provide a new, convenient marker of DNA-replicating cells; (f) reveal whether H2AX phosphorylation can serve as a marker of DNA repair, and (g) adapt the laser scanning cytometer (LSC) to quantify the number of DSBs per nucleus. (2) Activation of serine (Ser) proteases during apoptosis. Using fluorochrome-labeled inhibitors of Ser proteases (FLISP) as affinity ligands to active Ser proteases we will: (a) further characterize the specificity of these ligands; (b) correlate activation of Ser proteases with activation of initiator and effector caspases in intrinsic and extrinsic models of apoptosis; and (c) test fluorochrome-tagged phosphonate inhibitors of Ser proteases as alternative markers of protease activation. (3) Activation of transglutaminase (TGase 2) during apoptosis. A new cytometric assay of TGase 2 activation will be used to: (a) characterize the differences in morphology and in other physical/cytochemical attributes measured by cytometry of cells that undergo apoptosis with vs without TGase 2 activation; and (b) define conditions/causes under which apoptosis occurs with vs without TGase 2 activation. (4) Develop microtransponders (MTPs) as a platform for measuring cell viability with the goal of creating a high-content anticancer drug screening assay. Towards this goal: (a) the optimal conditions for growing cells attached to MTPs will be determined and cell growth and viability on these surfaces (by differential measurement of DNA and RNA content) will be compared with cell growth on standard solid-phase support; and (b) the ability to measure the cytostatic/cytotoxic effects of antitumor drugs on tumor cell lines will be tested and compared with the standard MTT cytotoxicity assay.